1. Mos Mediates the Mitotic Activation of p42 MAPK in Xenopus Egg Extracts 
Jianbo Yue, James E. Ferrell 
Current Biology 
Volume 14, Issue 17, Pages (September 2004)
DOI: /j.cub

2. Figure 1
Activation of a MEKK Activity in Δ90-Cyclin B-Treated Xenopus Egg Extracts
(A) Time course of Cdc2 activation, MEK phosphorylation, and p42 MAPK phosphorylation in Δ90-cyclin B-treated extracts. Cdc2 activity was assessed by histone H1 kinase assay and autoradiography. MEK phosphorylation was assessed by phospho-MEK immunoblotting. p42 MAPK phosphorylation was assessed by immunoblotting. The electrophoretically retarded MAPK band represents phosphorylated p42 MAPK.
(B) Activation of a MEKK activity in Δ90-cyclin B-treated extracts. Various volumes of extract were treated with or without Δ90-cyclin B for 60 min. Extracts were then incubated with recombinant GST-MEK1-Flag for 10 min. The phosphorylation of GST-MEK1-Flag was then assessed by phospho-MEK immunoblotting and densitometry. Equal loading of GST-MEK1-Flag was verified by MEK immunoblotting. (C) Quantitation of the data shown in (B).
(D) Dephosphorylation of 32P-labeled GST-MEK1-Flag in interphase and Δ90-cyclin B-treated extracts.
(E) Cumulative data from five dephosphorylation experiments. Points represent means ± standard error.
Current Biology  , DOI: ( /j.cub )

3. Figure 2 Fractionation of MEKK Activities
(A) Q-Sepharose chromatography. Interphase and M phase egg extracts were fractioned by Q-Sepharose chromatography. Aliquots of each fraction were incubated with recombinant GST-MEK1-Flag. Phosphorylated GST-MEK1-Flag was detected by phospho-MEK immunoblotting. Two peaks of activity were seen in the M phase Δ90-cyclin B-treated extracts.
(B) Gel filtration chromatography. MEKK activity and Mos protein coeluted with an apparent molecular weight of approximately 40 kDa.
(C) Immunoprecipitation of the partially purified MEKK activity with Mos antibodies. Aliquots (50 μl) of Superose 6 fraction 14 (panel B) were subjected to immunoprecipitation with Mos antibodies or control IgG. Portions of the resulting supernatants (supe) and pellets were incubated with GST-MEK1-Flag and MgATP, then immunoblotted with phospho-MEK antibodies.
Current Biology  , DOI: ( /j.cub )

4. Figure 3
Mos Degradation after Fertilization and in Cycling Egg Extracts
(A) Degradation after fertilization. Eggs were fertilized in vitro, dejellied, and subjected to immunoblotting for Mos (top) and phospho-MAPK (bottom). Five embryos (Mos) or two embryos (phospho-MAPK) were loaded per gel lane. Cleavages occurred at 85 and 115 min. Embryo drawings are adapted from [27].
(B) Degradation in cycling egg extracts. Extracts were prepared after eggs were treated for 2 min with ionophore A23187 (0.8 μM). At time zero, 35S-labeled in vitro-translated Mos was added. Taking aliquots every 5–10 min allowed assessment of residual Mos (by PhosphorImaging), p42 MAPK phosphorylation (by phospho-MAPK immunoblotting), and histone H1 kinase activity. Sperm morphology was used for monitoring cell cycle progression. Nuclear envelope breakdown was taken as the start of mitosis, and nuclear envelope reformation was taken as the end of mitosis. The panel on the right shows cumulative data from three independent experiments. Points represent means ± standard error.
Current Biology  , DOI: ( /j.cub )

5. Figure 4 Depletion of Mos from Crude Xenopus Egg Extracts
(A) p42 MAPK phosphorylation in control and Δ90-cyclin B-stimulated extracts that were mock depleted, depleted of Mos, or depleted of Mos and supplemented with bacterially expressed MBP-Mos (10 nM).
(B) Depletion of B-Raf and Raf-1 does not block p42 MAPK activation.
(C) Mos depletion blocks the transient p42 MAPK activation that occurs in cycling Xenopus egg extracts. Mitotic entry and exit were assessed from sperm morphology, as described for Figure 3B.
Current Biology  , DOI: ( /j.cub )